The long-range goal of this research program is the development of efficient in vivo gene delivery systems. The goal of this specific project is the development of an improved electro transfer system for the delivery of plasmid DNA to the skin. Skin is easily accessibility which makes it an excellent target for gene therapy applications whether it is for directly treating cutaneous diseases or utilizing the skin as a depot for delivering proteins directly to the circulation for systemic therapy. Electro transfer of skin is a simple, direct, in vivo method to deliver genes for therapy and can be accomplished in a minimally invasive way. We have previously developed devices and protocols to utilize electro transfer to effectively deliver plasmid DNA to the skin. We successfully demonstrated the utility of this approach for delivery of DNA vaccines, wound healing and ischemia. There were however still some shortcomings. One major issue of the current approach is that expression is confined to the epithelial layer. In addition, the applied voltages needed to achieve delivery on some occasions caused cellular or tissue damage. It is critical to address these issues in order to effectively translate this approach as well as make this approach feasible for other therapeutic applications particularly those that require increased serum levels of the expressed transgene. To address these issues we propose to combine electro transfer with the application of controlled moderate heat that is applied from an exogenous source. Raising the temperature of the skin to 43oC would increase the fluidity of the cell membranes as well as decrease the conductance within the skin. This will allow for delivery of plasmid DNA at lower voltages and also enable us to successfully delivery to deeper layers of the skin. We hypothesize that if electro transfer pulses are applied within a tissue at a moderately elevated temperature then it will be possible to achieve delivery at lower applied electric fields which would decrease the potential for damage and/or discomfort. We further hypothesize that if a combination of controlled exogenous heat and electro transfer is used then deeper penetration of the fields will be achieved thereby obtaining expression in the deeper layers of the skin. The following specific aims will be performed as part of this project. 1) to evaluate the combination of exogenous controlled heat and applied electric fields for delivery of plasmid DNA to the skin and to determine the duration of maximal expression levels and to determine if this time can be increased by performing multiple delivery procedures; 2) to determine the ability to control and/or target the delivery within the skin. Work will be conducted in a guinea pig model and will include evaluating depth of expression within the skin; and 3) to determine if the system established in the first two aims can deliver plasmids coding for proteins applicable for therapeutic purposes. The investigators have pioneered the use of electroporation for in vivo delivery so are well suited to successfully complete the study.